1.1 Field of the Invention
The present invention relates generally to transgenic plants having insecticidal capabilities, and to DNA constructs utilized to transfer genes conferring insect resistance into plant genomes. More specifically, the present invention relates to a method of expressing insecticidal proteins in plants transformed with a B. thuringiensis xcex4-endotoxin encoding gene, resulting in effective control of susceptible target pests.
1.2 Description of Related Art
1.2.1 Methods of Controlling Insect Infestation in Plants
The Gram-positive soil bacterium B. thuringiensis is well known for its production of proteinaceous parasporal crystals, or xcex4-endotoxins, that are toxic to a variety of Lepidopteran, Coleopteran, and Dipteran larvae. B. thuringiensis produces crystal proteins during sporulation which are specifically toxic to certain species of insects. Many different strains of B. thuringiensis have been shown to produce insecticidal crystal proteins. Compositions comprising B. thuringiensis strains which produce proteins having insecticidal activity have been used commercially as environmentally-acceptable topical insecticides because of their toxicity to the specific target insect, and non-toxicity to plants and other non-targeted organisms.
xcex4-endotoxin crystals are toxic to insect larvae by ingestion. Solubilization of the crystal in the midgut of the insect releases the protoxin form of the xcex4-endotoxin which, in most instances, is subsequently processed to an active toxin by midgut protease. The activated toxins recognize and bind to the brush-border of the insect midgut epithelium through receptor proteins. Several putative crystal protein receptors have been isolated from certain insect larvae (Knight et al., 1995; Gill et al., 1995; Masson et al., 1995). The binding of active toxins is followed by intercalation and aggregation of toxin molecules to form pores within the midgut epithelium. This process leads to osmotic imbalance, swelling, lysis of the cells lining the midgut epithelium, and eventual larvae mortality.
1.2.2 Transgenic B. thuringiensis xcex4-Endotoxins as Biopesticides
Plant resistance and biological control are central tactics of control in the majority of insecticide improvement programs applied to the most diverse crops. With the advent of molecular genetic techniques, various xcex4-endotoxin genes have been isolated and their DNA sequences determined. These genes have been used to construct certain genetically engineered B. thuringiensis products that have been approved for commercial use. Recent developments have seen new xcex4-endotoxin delivery systems developed, including plants that contain and express genetically engineered xcex4-endotoxin genes. Expression of B. thuringiensis xcex4-endotoxins in plants holds the potential for effective management of plant pests so long as certain problems can be overcome. These problems include the development of insect resistance to the particular Cry protein expressed in the plant, and development of morphologically abnormal plants because of the presence of the transgene.
Expression of B. thuringiensis xcex4-endotoxins in transgenic cotton, corn, and potatoes has proven to be an effective means of controlling agriculturally important insect pests (Perlak et al., 1990; Koziel et al., 1993; Perlak et al., 1993). Transgenic crops expressing B. thuringiensis xcex4-endotoxins enable growers to significantly reduce the application of costly, toxic, and sometimes ineffective topical chemical insecticides. Use of transgenes encoding B. thuringiensis xcex4-endotoxins is particularly advantageous when insertion of the transgene has no negative effect on the yield of desired product from the transformed plants. Yields from crop plants expressing certain B. thuringiensis xcex4-endotoxins such as Cry1A or Cry3A have been observed to be equivalent or better than otherwise similar non-transgenic commercial plant varieties. This indicates that expression of some B. thuringiensis xcex4-endotoxins does not have a significant negative impact on plant growth or development. This is not the case, however, for all B. thuringiensis xcex4-endotoxins that may be used to transform plants.
The use of topical B. thuringiensis-derived insecticides may also result in the development of insect strains resistant to the insecticides. Resistance to Cry1A B. thuringiensis xcex4-endotoxins applied as foliar sprays has evolved in at least one well documented instance (Shelton et al., 1993). It is expected that insects may similarly evolve resistance to B. thuringiensis xcex4-endotoxins expressed in transgenic plants. Such resistance, should it become widespread, would clearly limit the commercial value of corn, cotton, potato, and other germplasm containing genes encoding B. thuringiensis xcex4-endotoxins. One possible way to both increase the effectiveness of the insecticide against target pests and to reduce the development of insecticide-resistant pests would be to ensure that transgenic crops express high levels of B. thuringiensis xcex4-endotoxins (McGaughey and Whalon, 1993; Roush, 1994).
In addition to producing a transgenic plant which expresses B. thuringiensis xcex4-endotoxins at high levels, commercially viable B. thuringiensis genes must satisfy several additional criteria. For instance, expression of these genes in transgenic crop plants must not reduce the vigor, viability or fertility of the plants, nor may it affect the normal morphology of the plants. Such detrimental effects have two undesired results: they may interfere with the recovery and propagation of transgenic plants; they may also impede the development of mature plants, or confer unacceptable agronomic characteristics.
There remains a need for compositions and methods useful in producing transgenic plants which express B. thuringiensis xcex4-endotoxins at levels high enough to effectively control target plant insect pests as well as prevent the development of insecticide-resistant pest strains. A method resulting in higher levels of expression of the B. thuringiensis xcex4-endotoxins will also provide the advantages of more frequent attainment of commercially viable transformed plant lines and more effective protection from infestation for the entire growing season.
There also remains a need for a method of increasing the level of expression of B. thuringiensis xcex4-endotoxins which does not simultaneously result in plant morphological changes that interfere with optimal growth and development of desired plant tissues. For example, the method of potentiating expression of the B. thuringiensis xcex4-endotoxins in corn should not result in a corn plant which cannot optimally develop for cultivation. Achievement of these goals such as high expression levels as well as recovery of morphologically normal plants has been elusive, and their pursuit has been ongoing and an important aspect of the long term value of insecticidal plant products.
Described are novel methods for expressing Cry2A B. thuringiensis xcex4-endotoxins which lack significant Dipteran inhibiting activity in transformed plants. This method advantageously results in both increased levels of expression of B. thuringiensis xcex4-endotoxins as well as a higher rate of recovery of morphologically-normal plants.
By achieving high rates of expression, the present invention addresses another limitation of the prior art: development of insect resistance. Specifically, the instant invention provides a superior strategy for the delay or elimination of the development of resistance to Cry1A xcex4-endotoxins, the B. thuringiensis proteins most commonly expressed by transgenic lines. The disclosed methods involve expression of the Cry2A class of B. thuringiensis xcex4-endotoxins and particularly those that lack Dipteran-inhibiting activity. B. thuringiensis xcex4-endotoxins of the Cry2A group have no significant homology to Cry1A-type xcex4-endotoxins and display distinct binding and pore-forming characteristics (English et al., 1994), and as such are expected to control insects that become resistant to, or that are not affected by, Cry1A xcex4-endotoxins (Hofte and Whiteley, 1989).
In preferred embodiments, the present invention provides an isolated and purified DNA construct comprising a Cry2A xcex4-endotoxin-encoding region localized to a plastid or chloroplast, or localized to a plant cell nuclear genome and operably linked to a region encoding a plastid transit peptide (PTP). Preferred DNA constructs of the present invention include those constructs that encode Cry2A xcex4-endotoxins lacking Dipteran-inhibitory activity, though complete inactivity towards Dipterans is not required. In an illustrative embodiment, DNA constructs of the present invention encode a Cry2Ab xcex4-endotoxin operably linked to a DNA segment (or sequence) encoding a plastid transit peptide, which is one means of enabling localization of a Cry2Ab xcex4-endotoxin to a plastid or chloroplast. In certain embodiments, the Cry2Ab xcex4-endotoxin comprises the sequence of SEQ ID NO:2. The inventors contemplate, however, that any Cry2A xcex4-endotoxin lacking Dipteran-inhibitory activity may be utilized according to the present invention, with those bearing substantial homologies to Cry2Ab being particularly preferred.
In another embodiment, the DNA constructs of the present invention exploit nucleic acid segments encoding PTPs to potentiate expression of the xcex4-endotoxin. The use of one type of PTP, a chloroplast targeting peptide (CTP), in conjunction with a crylA B. thuringiensis transgene to promote expression of the transgene in the transformed plant is disclosed in U.S. Pat. No. 5,500,365 (specifically incorporated herein by reference in its entirety). Where increased expression was observed, however, it was ascribed in part to the use of a new 5xe2x80x2 untranslated leader sequence in the expression vector.
In contrast to the prior art, the present invention discloses a structural DNA sequence that causes the production of an RNA sequence which encodes a targeted fusion protein comprising an amino-terminal plastid transit peptide with a Cry2Ab xcex4-endotoxin; and a 3xe2x80x2 non-translated DNA sequence which functions in plant cells to cause transcriptional termination and the addition of polyadenylated nucleotides to the 3xe2x80x2 end of the RNA sequence. Surprisingly, this DNA construct results in increased levels of expression of the Cry2A xcex4-endotoxin. The targeted fusion protein is non-active to all species, but is produced as a means for localizing the mature, insecticidally active xcex4-endotoxin protein to the chloroplast, yielding surprising and unexpected beneficial agronomic effects.
One embodiment conceived of in the present invention is the introduction of a gene encoding a Cry2A xcex4-endotoxin lacking Dipteran activity into the chloroplast or plastid genome. Alternatively, a gene encoding a Cry2A xcex4-endotoxin lacking Dipteran activity could be expressed from an autonomously replicating episomal element located within the chloroplast or plastid.
In another preferred embodiment, the invention provides for transgenic plants which have been transformed with an isolated and purified DNA construct that is translated and expressed at high levels by the plant. Both monocot and dicot plants may be transformed according to the methods and with the DNA constructs disclosed herein. The plant transformed by the instant invention may be prepared, in a further preferred embodiment, by a process including obtainment of the isolated and purified DNA construct, and then transforming the plant with the construct so that the plant expresses the proteins for which the construct encodes. The inventors have observed that transformation of plants by the disclosed methods results in increased frequency of transformants which express the transgene, as well as the generation of more morphologically normal plants from initial transformants.
It is contemplated that the increased expression levels observed in the disclosed invention will allow for reduced development of insect resistance to Bt xcex4-endotoxins. This may be achieved by transforming a plant with the preferred DNA construct to achieve high rates of Cry2A expression alone, or by simultaneously exposing target insects to Cry1A and non-Dipteran active Cry2A xcex4-endotoxins expressed in susceptible plants. Such insects include Ostrina spp., Diatraea spp., Helicoverpa spp., and Spodoptera spp., in Zea mays; Heliothis virescens, Helicoverpa spp., Pectinophora spp., in Gossypium hirsutum; Anticarsia spp., Pseudoplusia spp., Epinotia spp., in Glycine max; and Scirpophaga incertulas in Oryza sativa. 
It is therefore contemplated that the method disclosed by the present invention will provide many advantages over the prior art including those specifically outlined above. These advantages include: obtaining improved control of susceptible insects; minimizing the development of insecticide-resistant insect strains; obtaining a greater number of commercially viable insect resistant plant lines; achieving season long protection from insect pathogens; and increasing the incidence of morphologically-normal transformed plants. An additional advantage of the present invention is that reduced numbers of transgenic lines would need to be produced in order to identify a transgenic event with normal growth characteristics.
2.1 Nucleic Acid Compositions
In one important embodiment, the invention provides an isolated and purified nucleic acid construct comprising a Cry2A coding region and a PTP coding region. These DNA constructs, when transferred into a plant, undergo cellular processes resulting in increased expression of xcex4-endotoxins in the transgenic plant. The Cry2A endotoxins of the instant invention are preferably not effective against Dipteran species, though some adverse effects on Dipterans may be tolerated. In certain embodiments, the DNA construct encodes a Dipteran-inactive Cry2Ab xcex4-endotoxin, and in more preferred embodiments, the Cry2Ab xcex4-endotoxin has the polypeptide sequence of SEQ ID NO:2, or one substantially homologous to the polypeptide sequence of SEQ ID NO:2. Such nucleotide homologues may be greater than approximately 88% homologous, greater than about 90% homologous, greater than about 95% homologous, and even greater than about 99% homologous with the Cry2Ab xcex4-endotoxin disclosed in SEQ ID NO:2. Exemplary peptides include those that are about 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or even 99 or greater percent homologous to the Cry2Ab xcex4-endotoxin disclosed in SEQ ID NO:2.
In even more preferred embodiments, the DNA construct of the present invention comprises a Cry2Ab xcex4-endotoxin-encoding region with the nucleic acid sequence of SEQ ID NO:1, or a sequence substantially homologous to that of SEQ ID NO:1. Also envisioned as within the scope of this invention are those DNA constructs having segments with substantial homologies to the nucleic acid sequence disclosed in SEQ ID NO:1, such as those which may be about 90% homologous, or about 95% homologous, or even about 99% homologous. More specifically, homologous nucleic acid sequences included in the present invention include those that are about 90, 91, 92 ,93, 94, 95, 96, 97, 98, and 99 percent homologous to the nucleic acid sequence of SEQ ID NO:1.
The DNA constructs provided herein also include a PTP coding region positioned upstream of the cry2A xcex4-endotoxin coding region and downstream of a promoter. These plastid transit peptide coding regions may encode any plant functional PTP, and may operate to target encoded proteins to certain plastids within the plant cell, or to increase the expression of the xcex4-endotoxin for which the DNA construct encodes. In preferred embodiments, the present invention may include a PTP selected from the group including zmSSU, PTP1, PTP1xcex94, and PTP2, or any other plant functional PTPs. More preferably, the plastid transit peptide coding region encodes a plastid transit peptide having the amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or any polypeptide sequence substantially homologous to these. Even more preferably, the instant invention comprises a plastid transit peptide coding region having the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9, or a nucleic acid sequence which is substantially homologous to these.
Also, the inventors contemplate that the present invention would further achieve the goals of increased pathogenicity to pests, and result in decreased development of pesticide-resistant insects, if the DNA constructs provided herein were co-expressed along with other pesticidal compositions such as other proteins. Accordingly, the invention provides for use of the disclosed DNA constructs which further comprise plant-expressible coding regions for other Cry proteins. Included in these would be coding regions for Cry1 proteins such as Cry1A, Cry1Ab, Cry1Bb, or Cry1 chimeras (see co-pending U.S. applications Ser. No.""s 08/754,490 and 08/922,505, and co-pending PCT Application PCT/US97/17507 based on U.S. application Ser. No. 08/721,259, each specifically incorporated herein by reference in its entirety).
In certain preferred embodiments, the DNA construct is an expression cassette which can be excised and isolated from said plasmid.
2.2 Additional Nucleic acid Composition Elements
The polynucleotide compositions of the present invention are useful in transforming both monocotyledonous and dicotyledonous plants. Accordingly, the DNA construct of the present invention may further comprise other various regulatory elements to aid in protein expression and to further facilitate introduction of the DNA construct into the plant. One example of this is the inclusion, in the DNA construct, of an intron positioned in the untranslated leader, upstream relative to the plastid transit peptide coding region. One useful leader sequence is the petunia heat shock protein. In various alternative embodiments, the intron may be any of the following: Adh intron 1, sucrose synthase intron, TMV omega element, maize heat shock protein (hsp) 70, or the rice ActI intron. In preferred embodiments, the intron is either maize heat shock protein 70 or petunia heat shock protein 70.
Provided in another preferred embodiment of the present invention is a polynucleotide sequence comprising a substantially Dipteran inactive cry2A xcex4-endotoxin coding region and a PTP coding region positioned under the control of a plant operable promoter. The use of a promoter is required for driving cellular processes so that expression of the gene is maximized. Preferred promoters include the following: CaMV 35S, histone, CaMV 19S, nos, OCS, Adh, sucrose synthase, xcex1-tubulin, actin, cab, PEPCase, ssRUBISCO, Actl, Famv, enhanced FMV, or R-gene complex associated promoters. In more preferred embodiments, the promoter is the enhanced or duplicated CaMV 35S promoter (Kay et al., 1987). In additional preferred embodiments, the promoter is the FMV35S promoter. Plant chloroplast or plastid functional promoters are also within the scope of the present invention.
The present invention further contemplates the inclusion of a terminator region in the DNA construct to aid cellular processes involved with protein expression. In various embodiments, this terminator may be any of the following: the Agrobacterium tumefaciens nopaline synthase gene terminator, the Agrobacterium tumefaciens octopine synthase gene terminator, and the 3xe2x80x2 end of the protease inhibitor I or II genes from potato or tomato. In an especially preferred embodiment, the terminator is the Agrobacterium tumefaciens nopaline synthase gene terminator.
2.3 Transformation Vectors
Because the DNA construct of the present invention is primarily, though not exclusively, intended for use in the transformation of plants, it is in certain preferred embodiments, contained within an expression vector. Such expression vectors may contain a variety of regulatory and other elements intended to allow for optimal expression of the desired proteins for which the expression vector encodes. These additional elements may include promoters, terminators, and introns as outlined above in section 2.2. The vector containing the DNA construct and any regulatory or other elements may be selected from the group consisting of a yeast artificial chromosome, bacterial artificial chromosome, a plasmid, or a cosmid.
Further, the expression vectors themselves may be of a variety of forms. These forms may differ for various reasons, and will likely be comprised of varying components depending upon whether they are intended to transform a monocotyledonous plant or a dicotyledonous plant. For example, FIG. 1 illustrates one possible embodiment, where the monocotyledonous expression vector contains the cry2Ab gene in the plasmid designated as (SEQ ID NO:16). It is further contemplated that other expression vectors containing the expression cassettes embodied in these plasmid vectors, as well as expression cassettes containing substantial homologues, will also be useful transformation constructs. Accordingly, any transformation vector containing the nucleic acid sequence of from nucleic acid 1781 to 5869 of SEQ ID NO:16.
FIG. 2 illustrates one possible dicotyledonous expression vector. It contains the cry2Ab gene embodied in the plasmids designated as pMON33827 (SEQ ID NO:13), pMON33828 (SEQ ID NO:14), and pMON33829 (SEQ ID NO:15). As with the illustrative monocotyledonous transformation vectors, the inventors further contemplate that other expression vectors containing the expression cassettes embodied in these plasmid vectors, or substantial homologues to those expression cassettes, will be useful as dicotyledonous transformation constructs. Preferred dicotyledonous expression cassettes include those embodied by nucleic acids 17 to 3182 of SEQ ID NO:13; nucleic acids 17 to 3092 of SEQ ID NO:14; and nucleic acids 17 to 3155 of SEQ ID NO:15. Illustrative embodiments of vectors containing such expression cassettes are disclosed in the sequences designated herein as SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.
Vectors further envisioned to be within the scope of the present invention include those vectors capable of containing both the Dipteran-inactive cry2A nucleic acid compositions disclosed in section 2.1 above, as well as any other DNA constructs which further comprise plant-expressible coding regions for other Cry proteins such as a Cry1 protein. Vectors capable of containing both of these constructs may further comprise an internal ribosome entry site between the DNA construct; they may also contain a variety of different cistrons, rendering them polycistronic or multicistronic
2.4 Transformed Host Cells
Another preferred embodiment of the present invention encompasses cells transformed with the DNA constructs disclosed herein in sections 2.1 and 2.2, and by use of the transformation vectors disclosed in section 2.3. Transformed cells contemplated in the present invention include both prokaryotic and eukaryotic cells which express the proteins encoded-for by the novel DNA constructs of the present invention. The process of producing transgenic cells is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operatively linked to a coding region that encodes a B. thuringiensis xcex4-endotoxin. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the xcex4-endotoxin in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular xcex4-endotoxin or endotoxins expressed in a particular transgenic cell, the invention also provides for the expression of xcex4-endotoxin antisense mRNA; intron antisense mRNA; PTP antisense mRNA; or UTR antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.
In a preferred embodiment, the invention encompasses a plant cell which has been transformed with a nucleic acid segment or DNA construct of the invention, and which expresses a gene or gene segment encoding one or more of the Dipteran-inactive Cry2A B. thuringiensis xcex4-endotoxins as disclosed herein. As used herein, the term xe2x80x9ctransgenic plant cellxe2x80x9d is intended to refer to a plant cell that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (xe2x80x9cexpressedxe2x80x9d), or any other genes or DNA sequences which one desires to introduce into the non-transformed plant, such as genes which may normally be present in the non-transformed plant but which one desires to either genetically engineer or to have altered expression.
It is contemplated that in some instances the genome of a transgenic plant of the present invention will have been augmented through the stable introduction of a Dipteran-inactive Cry2A B. thuringiensis xcex4-endotoxin-encoding DNA constructs as disclosed in sections 2.1 and 2.2 above. In some instances, more than one transgene will be incorporated into the nuclear genome, or into the chloroplast or plastid genome of the transformed host plant cell. Such is the case when more than one crystal protein-encoding DNA segment is incorporated into the genome of such a plant. In certain situations, it may be desirable to have one, two, three, four, or even more B. thuringiensis crystal protein-encoding polynucleotides (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic plant.
In preferred embodiments, the introduction of the transgene into the genome of the plant cell results in a stable integration wherein the offspring of such plants also contain a copy of the transgene in their genome. The heritability of this genetic element by the progeny of the plant into which the gene was originally introduced is a preferred aspect of this invention. A preferred gene which may be introduced includes, for example a B. thuringiensis xcex4-endotoxin, and particularly one or more of those described herein.
Means for transforming a plant cell and the preparation of a transgenic cell line are well-known in the art (as exemplified in U.S. Pat. Nos. 5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341, all specifically incorporated herein by reference in their entirety), and are briefly discussed herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed crystal proteins. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified crystal protein, which will be expressed in the resultant recombinant cells, and/or which will impart an improved phenotype to the regenerated plant.
Transgenic cells specifically contemplated in the present invention include transgenic plant cells. Particularly preferred plant cells include those cells obtained from corn, wheat, soybean, turf grasses, ornamental plant, fruit tree, shrubs, vegetables, grains, legumes, and the like, or any plant into which introduction of a Dipteran-inactive B. thuringiensis xcex4-endotoxin transgene is desired.
2.5 Transformed Plants
In another aspect, plants transformed with any DNA construct of the present invention that express the proteins for which the construct encodes, are contemplated as being a part of this invention. Accordingly, the invention further provides transgenic plants which have been transformed with a DNA construct, as disclosed herein in sections 2.1 and 2.2, and transformed by use of transformation vectors as disclosed in section 2.3. Agronomic, horticultural, ornamental, and other economically or commercially useful plants can be made in accordance with the methods described herein, to express B. thuringiensis xcex4-endotoxins at levels high enough to confer resistance to insect pathogens while remaining morphologically normal.
Such plants may co-express the xcex4-endotoxin polypeptide along with other antifungal, antibacterial, or antiviral pathogenesis-related peptides, polypeptides, or proteins; insecticidal proteins; proteins conferring herbicide resistance; and proteins involved in improving the quality or quantity of plant products or agronomic performance of plants. Simultaneous co-expression of multiple proteins in plants is advantageous in that it exploits more than one mode of action to control plant pathogenic damage. This can minimize the possibility of developing resistant pathogen strains, broaden the scope of resistance, and potentially result in a synergistic insecticidal effect, thereby enhancing a plant""s ability to resist insect infestation (Intl. Patent Appl. Publ. No. WO 92/17591, Oct. 15, 1992, specifically incorporated herein by reference in its entirety).
The transformed plant of the current invention may be either a monocotyledonous plant or a dicotyledonous plant. Where the plant is a monocotyledonous plant, it may be any one of a variety of species. Preferred monocotyledonous species encompassed by the present invention may include maize, rice, wheat, barley, oats, rye, millet, sorghum, sugarcane, asparagus, turfgrass, or any of a number of other grains or cereal plants. In preferred embodiments, the monocot is a maize plant.
The present invention also contemplates a variety of dicotyledonous plants such as cotton, soybean, tomato, potato, citrus, tobacco, sugar beet, alfalfa, fava bean, pea, bean, apple, cherry, pear, strawberry, raspberry, or any other legume, tuber, or fruit plant. In preferred embodiments, the dicot is a soybean plant, a tobacco plant, or a cotton plant.
Many of the plants intended to be transformed according to the disclosed invention are commercial crop plants. The commercial form of these plants may be the original plants, or their offspring which have inherited desired transgenes. Accordingly, plants further contemplated within the ambit of the present invention include any offspring of plants transformed with any of the permutations of the DNA construct which are noted in this application. Specifically, the offspring may be defined as an R0 transgenic plant. Other progeny of the transformed plant are also included within the scope of the present invention, including any progeny plant of any generation of the transformed plant, wherein the progeny plant has inherited the DNA construct from any R0 plant.
Upon transformation with a specific DNA construct, the nucleic acid or polynucleotide segments of the construct may be incorporated in various portions into a chromosome of the transformant. Therefore, in another embodiment, the present invention encompasses any transgenic plant or plant cell prepared by the use of a DNA construct disclosed herein. Such a plant or cell encompassed by the present invention includes those prepared by a process which has the following steps: (1) obtaining a DNA construct including a Dipteran-inactive Cry2A B. thuringiensis xcex4-endotoxin coding region positioned in frame and under the control of a promoter operable in the plant, and a plastid transit peptide coding region positioned upstream of the Cry2A B. thuringiensis xcex4-endotoxin coding region and downstream of the promoter; and (2) transforming the plant with the obtained DNA construct, so that the plant expresses the Cry2A B. thuringiensis xcex4-endotoxin. The plant may also have been transformed so that it further incorporates into its genome and expresses other Cry xcex4-endotoxins.
In a related aspect, the present invention also encompasses a seed produced by the transformed plant, a progeny from such seed, and a seed produced by the progeny of the original transgenic plant, produced in accordance with the above process. Such progeny and seeds will have a Dipteran-inactive B. thuringiensis xcex4-endotoxin transgene stably incorporated into its genome, and such progeny plants will inherit the traits afforded by the introduction of a stable transgene in Mendelian fashion. All such transgenic plants having incorporated into their genome transgenic DNA segments encoding any DNA construct disclosed herein, particularly those disclosed in sections 2.1 and 2.2 are aspects of this invention.
Recombinant plants, cells, seeds, and other tissues could also be produced in which only the mitochondrial or chloroplast DNA has been altered to incorporate the molecules envisioned in this application. Promoters which function in chloroplasts have been known in the art (Hanley-Bowden et al., Trends in Biochemical Sciences 12:67-70, 1987). Methods and compositions for obtaining cells containing chloroplasts into which heterologous DNA has been inserted has been described by Daniell et al., U.S. Pat. No. 5,693,507 (1997).
2.6 Plant Transformation Methods
2.6.1 Method of Expressing a Cry2A xcex4-Endotoxin in a Plant
In another preferred embodiment, the present invention provides a method for expressing Dipteran-inactive Cry2A B. thuringiensis xcex4-endotoxins at high levels in transgenic plants. The disclosed methods may exploit any of the DNA constructs disclosed in sections 2.1 and 2.2 above, as well as any of the transformation vectors disclosed, for example, in section 2.3 above. The contemplated methods enable Cry2A xcex4-endotoxins, an alternative to Cry1A B. thuringiensis xcex4-endotoxins for the control of several insect pests, to be expressed in plants without negatively affecting the recovery of agronomic qualities of transgenic plants. The invention described herein also enables expression of Cry2A xcex4-endotoxins at levels up to 25 times higher than that achieved by current methods.
The method described here thus enables plants expressing Cry2A to be used as either an alternative or supplement to plants expressing Cry1A-type B. thuringiensis xcex4-endotoxins for both control and resistance management of key insect pests, including Ostrina sp, Diatraea sp, Helicoverpa sp, Spodoptera sp in Zea mays; Heliothis virescens, Helicoverpa sp, Pectinophora sp. in Gossypium hirsutum; and Anticarsia sp, Pseudoplusia sp, Epinotia sp in Glycine max. It is also contemplated that the methods described may be used to dramatically increase expression of B. thuringiensis xcex4-endotoxins including and related to Cry2A, thus increasing its effectiveness against target pests and decreasing the likelihood of evolved resistance to these proteins. In one embodiment of the present invention, the Cry2Ab xcex4-endotoxin is expressed. Target pests of this protein and their common hosts are shown below in Table 1.
The method of expressing a Cry2A B. thuringiensis xcex4-endotoxin in a plant disclosed herein includes the steps of: (1) obtaining nucleic acid segment comprising a promoter operably linked to a first polynucleotide sequence encoding a plastid transit peptide, and a second polynucleotide sequence, encoding a Cry2A B. thuringiensis xcex4-endotoxin lacking Dipteran activity, to yield a fusion protein comprised of an amino-terminal plastid transit peptide and a Cry2A B. thuringiensis xcex4-endotoxin lacking Dipteran activity; and (2) transforming the plant with the DNA construct of step 1 so that the plant expresses the protein fusion. In a preferred embodiment, the nucleic acid segment employed in step (1) of this method is structured so that the 5xe2x80x2 end of the second polynucleotide sequence is operably linked in the same translational reading frame to the 3xe2x80x2 end of the first polynucleotide sequence.
The plant or plant cell transformed by the method disclosed herein may be either a monocotyledonous plant or a dicotyledonous plant. Where the plant is a monocotyledonous plant, it may be any one of a variety of species. Preferred monocotyledonous species encompassed by the present invention may include maize, rice, wheat, barley, oats, rye, millet, sorghum, sugarcane, asparagus, turfgrass, or any of a number of other grains or cereal plants. In preferred embodiments, the monocot is a maize plant.
The present invention also contemplates a process by which a variety of dicotyledonous plants or plant cells are transformed. Such dicotyledonous plants may include plants such as cotton, soybean, tomato, potato, citrus, tobacco, sugar beet, alfalfa, fava bean, pea, bean, apple, cherry, pear, strawberry, raspberry, or any other legume, tuber, or fruit plant. In preferred embodiments, the dicot is a soybean plant, a tobacco plant or cell, or a cotton plant or cell.
2.6.2 Method of expressing a Cry2Ab xcex4-endotoxin in a Progeny Plant
As noted with regard to other embodiments disclosed in the present invention, many of the plants intended to be transformed according to the disclosed invention are commercial crop plants. The commercial form of these plants may be the original plants, or their offspring which have inherited desired transgenes. Accordingly, the inventors further contemplate that the method disclosed herein includes a method of producing a transgenic progeny plant or progeny plant cell. The method of producing such progeny includes: The method of expressing a Cry2A B. thuringiensis xcex4-endotoxin in a plant disclosed herein includes the steps of: (1) obtaining nucleic acid segment comprising a promoter operably linked to a first polynucleotide sequence encoding a plastid transit peptide, and a second polynucleotide sequence, encoding a Cry2A B. thuringiensis xcex4-endotoxin lacking Dipteran activity, to yield a fusion protein comprised of an amino-terminal plastid transit peptide and a Cry2A B. thuringiensis xcex4-endotoxin lacking Dipteran activity; (2) obtaining a second plant; and (3) crossing the first and second plants to obtain a crossed transgenic progeny plant or plant cell which has inherited the nucleic acid segments from the first plant. The present invention specifically encompasses the progeny, progeny plant or seed from any of the monocotyledonous or dicotyledonous plants, including those noted in sections 2.5 and 2.6.1 above.
2.6.3 Method of Co-Expressing Cry2Ab and other Cry B. thuringiensis xcex4-endotoxins in a Plant and a Progeny Plant
In another preferred embodiment, the method of expressing the Dipteran-inactive Cry2A B. thuringiensis xcex4-endotoxin disclosed herein includes co-expression of the disclosed DNA construct in any of its various embodiments, along with a Cry1 B. thuringiensis xcex4-endotoxin. The method of expressing these Cry B. thuringiensis xcex4-endotoxins together is expected to achieve increased insecticidal properties in the transformed plant through increased expression and decreased development of insect resistance - all of which are desired results not present in existing technologies. This co-expression may be in the original transformant, or in any number of generations of progeny of the original transformant which have inherited the genes to co-express the proteins encoded for by any of the DNA constructs disclosed herein.